The Study Of Pirfenidone In The Treatment Of Hepatic Fibrosis By Inhibiting Autophagy Of Hepatic Stellate Cells And Promoting Apoptosis

Objective:To evaluate the effect of pirfenidone（PFD）on rat model of hepatic fibrosis,and to explore the effect of PFD on proliferation,migration and apoptosis of hepatic stellate cells through autophagy pathway.Methods:In vitro experiment,rat hepatic stellate cells（HSC T6）were treated with PFD at0,2,4,6 and 8 mmol/L for 48 h,and the cell viability was measured by CCK-8method,and then the final concentration of PFD at 0,2.5,5 and 7.5 mmol/L was selected for the follow-up study.The expression ofα-smooth muscle actin（α-SMA）,collagen-Ⅰ,heat shock protein 47（HSP-47）,microtubule-associated proteins light chain 3（LC3）,autophagy-related gene 6（Beclin1）,sequestosome 1（P62）,B-cell lymphoma-2 protein（Bcl-2）and Bcl-2 associated x Drotein（BAX）were measured by Western Blot（WB）.HSC T6 cells were divided into control,PFD（5 mmol/L）,RA（100 nmol/L）and PFD+RA groups.After 48h of PFD and RA treatment,the migration ability of cells in each group was measured by cell scratch method,the apoptosis of cells in each group was measured by flow cytometry,and the expression ofα-SMA,collagen-Ⅰ,HSP-47,LC3,Beclin1,P62,Bcl-2 and BAX were measured by WB.One-way ANOVA was used to compare between groups,and Dunnet t test or LSD was used for multiple comparisons.In vivo experiment,SD rats with hepatic fibrosis induced by carbon tetrachloride were randomly divided into normal group,fibrosis group and PFD group.Rats in fibrosis group and PFD group were fed with 1:1 ratio of CCl₄to olive oil,and each rat was intraperitoneally injected with 1 m L/kg twice a week for 5 weeks,and normal saline was injected with the same dosage and method as those in fibrosis group and PFD group,after 4 weeks,the fibrosis group was not treated,and the PFD group was given 1 m L/kg/d by oral gavage.At the end of the 9th week,the livers of all rats were collected,and the protein expressions ofα-SMA,collagen-Ⅰ,HSP-47,LC3,Beclin1and P62 in normal,fibrotic and PFD groups were detected by HE,Masson,Sirius and WB.One-way ANOVA was used to compare between groups,and Dunnet t test or LSD was used for multiple comparisons.Results:HSC T6 cells were treated with 0,2,4,6,8 mmol/L PFD.The results showed that with the increasing of PFD concentration,the viability of HSC T6 cells was significantly inhibited,and the half-inhibitory concentration was 5 mmol/L.Therefore,the concentrations of 0,2.5,5,7.5 mmol/L PFD were selected for the follow-up study.Cell scratch and cloning experiments showed that with the increasing of PFD concentration,the viability and migration ability of HSC decreased gradually,and the number of clonal colonies decreased gradually.Flow cytometry showed that with the increasing of PFD concentration,the apoptosis rate of HSC increased.WB showed that with the increasing of PFD concentration,the expression ofα-SMA,collagen-Ⅰand HSP-47 decreased gradually,the expression of autophagy-related protein P62 increased gradually,the expression of Beclin1 decreased gradually,the transformation rate of LC3-Ⅱ/LC3-Ⅰdecreased gradually,the expression of apoptosis related protein BAX increased gradually,the expression of Bcl-2 decreased gradually.HSC T6 cells were treated with RA and PFD.Cell scratch test showed that the migration ability of HSC T6 cells was the highest in RA group,and was significantly higher in combined group than in PFD group（P<0.05）.Flow cytometry showed that the apoptosis rate of RA group was the lowest in RA group,and was significantly lower in combined group than in PFD group（P<0.05）.WB showed that the expression ofα-SMA,collagen-Ⅰand HSP-47 in RA group was significantly higher than in control group,but there was no statistical difference（P>0.05）,the expression of hepatic fibrosis proteins in combined group was significantly higher than in PFD group（P<0.05）.Autophagy-Related protein showed that the expression of P62 protein was the lowest in RA group,the expression of Beclin1 protein was the highest in LC3-Ⅱ/LC3-Ⅰ,the expression of P62 protein was the lowest in RA group,and was significantly higher in Beclin1 protein than in PFD group（P<0.05）.Bcl-2 protein showed that the expression of BAX protein was the lowest in RA group,the expression of Bcl-2 protein was the highest in RA group,the expression of BAX protein was the lowest in PFD group,and was significantly higher in Bcl-2 protein than in PFD group（P<0.05）.HE,Masson and Sirius red staining showed that the structure of liver in normal group was clear,the hepatic lobules were arranged regularly,the hepatocytes were arranged radially around the central vein,there was no deposition of collagen fibril,the volume of hepatocytes in fibrosis group was larger than that in normal group,the deposition of collagen fibril could be seen in some areas in fibrosis group,the deposition of collagen fibril in PFD group was significantly improved.WB results showed that the expression ofα-SMA,HSP-47 and collagen-Ⅰwas significantly higher in fibrosis group than that in normal group（P<0.05）,the expression of P62protein was not significantly different in autophagy-related protein,the expression of Beclin1 protein and the transformation of LC3-Ⅱ/LC3-Ⅰwere significantly higher in fibrosis group（P<0.05）.However,the expression ofα-SMA,HSP-47 and collagen-Ⅰwas significantly lower in fibrosis group（P<0.05）,the expression of P62 protein was significantly higher in autophagy-related protein,the expression of Beclin1 protein and the transformation of LC3-Ⅱ/LC3-Ⅰwere significantly lower in fibrosis group（P<0.05）.Conclusions:（1）After PFD treatment,HSC T6 cells exhibited inhibition of cell proliferation,decreased migration ability and activity,impaired autophagy pathway and increased apoptosis.（2）PFD may inhibit autophagy of HSC T6 cells,reduce the cell activity of HSC T6 cells,and promote apoptosis.（3）PFD could reduce the level of liver fibrosis in rats,and inhibit autophagy in liver fibrosis rats.